Project Summary Pancreatic cancer is a uniformly lethal form of cancer with patients rarely surviving two years after diagnosis due to pronounced metastasis, immune suppression and evasion, desmoplasia, and unchecked tumor proliferation that together confer therapeutic resistance. Cancer metastatic potential is mediated by the G protein-coupled chemokine receptor CXCR4. The chemokine CXCL12 is the cognate ligand for CXCR4 and is an immune mediator produced by both stromal cells in the tumor microenvironment and normal cells at metastatic destinations. While a wealth of reports attribute elevated CXCR4 expression with pro-tumorigenic effects in numerous cancers, the precise mechanistic roles for CXCR4 and CXCL12 in non-metastatic pathways of pancreatic cancer progression remain poorly understood. Our published and exciting preliminary data have revealed a novel mechanism whereby CXCL12 can either promote or inhibit tumor progression and metastasis based on its ability to activate CXCR4 as a monomer or dimer. In pursuit of determining the mechanism behind CXCR4-mediated cancer progression, we have engineered a locked monomer variant of CXCL12 that acts as a balanced agonist at CXCR4, activating the entirety of its G protein and ?-arrestin signaling repertoire, as well as a locked dimer biased agonist variant which activates only a subset. We hypothesize that biased signaling at CXCR4 prevents tumor migration and progression while attracting cytotoxic T lymphocytes from the bone marrow and tumor periphery to infiltrate the tumor and kill cancer cells. The overall goal of this proposal is to harness biased agonist signaling as a multi-pronged anti-tumor approach to abrogate pancreatic cancer progression. Aim 1 will use genetically engineered mouse models of pancreatic cancer to analyze the in vivo influence of biased agonist signaling in tumor progression. Aim 2 will use transgenic mouse models and cell culture approaches to investigate the sparsely-studied effects of biased signaling on immune cell trafficking, infiltration, and tumor cell killing in the tumor microenvironment. Aim 3 will delve into the amino-acid level interactions between CXCL12 agonists with CXCR4 receptor to determine the mechanisms responsible for anti-tumor ligand biased and tissue biased signaling. The overall impact of the proposed work is that we will reveal a targetable structural and biochemical biased agonist signaling mechanism that explains the pleiotropic effects of CXCL12 and CXCR4 in cancer.